This grant application concerns two topics. The first concerns the role of secreted cell death factors in Bcr-Abl positive chronic myelogenous leukemia (CML). We will primarily focus on 24p3, a lipocalin that is secreted by IL-3 deprived mouse 32D cells, and was demonstrated by another group to cause cell death in many types of hematopoietic cells. Lipocalins are a large family of secreted proteins that function as ligand-binding molecules with a variety of biological activities. We found that Bcr-Abl positive mouse 32D cells express high level of 24p3 transcripts in the absence or presence of IL-3. Conditioned medium from Bcr-Abl positive 32D cells induces cell death in normal mouse marrow cells but not in Bcr-Abl positive cells. We hypothesize that Bcr-Abl induced leukemia results from two separate effects: 1) the oncogenic immortalization of leukemia cells; and 2) the induction of cell death factors that destroy normal blood cells, leading to immuno-suppression and other effects that affect normal blood cell function. We plan to characterize the 24p3 protein secreted by Bcr-Abl and investigate the mechanisms of 24p3 induction and the resistance of Bcr-Abl cells to 24p3-mediated cell death. We also plan to determine the effects of reduced levels of 24p3 secretion on the induction of leukemia in mouse models using lentivirus mediated transduction of anti-sense 24p3. The second topic concerns the inhibitory role of Bcr directed towards Bcr-Abl positive cells and, particularly, in cells resistant to Gleevec (STI-571). We will search for more potent forms of the Bcr inhibitory structure and study the mechanism of action. We have developed an efficient lentivirus gene transduction system, which is very useful for gene transduction in hematopietic cells. We will transduce cells with the replication-defective BCR encoding lentivirus equipped with a bi-cistronic message, which allows expression of BCR and a GFP reporter gene. Both in vitro cell systems and mouse models for CML will be tested with the BCR lentivirus to determine the effects of BCR expression in this leukemia. Based on our published papers, we propose a hypothesis that endogenous Bcr down-regulates the Bcr-Abl oncoprotein and thus reduction of Bcr protein levels will enhance the intrinsic oncogenic activity of the Bcr-Abl oncoprotein. We will test this hypothesis by expressing 3' anti-sense BCR sequences and the ABL SH2 domain sequence in Bcr-Abl positive cells.